The present invention relates to genes encoding regulators of gibberellic acid biosynthesis in plants. Plant development is affected by alterations in the nature or quantity of expression products of these genes. A family of genes, found in monocotyledonous plants (monocots), codes for a composition essential for the conversion of GGPP to ent-kaurene involved in the early steps of gibberellic acid (GA) biosynthesis. An illustrative member of the family, the gens Anther earl (An1), is identified in maize and cloned, and its functional attributes are characterized.
That GA is important in plant development is illustrated by the correlation between increased vigor in hybrid maize and higher GA levels compared to parental levels, and the greater response of inbreds (compared to hybrids) to exogenously applied GA content (Rood et al., 1988). Further, RFLP analysis points to known GA biosynthetic loci as quantitative trait loci (QTLs) for height in maize hybrids (Beavis et al., 1991), suggesting a role for GA in heterosis. The importance of GA in plant development is further evidenced in the phenotype of GA-deficient mutants of maize, which includes: reduced plant stature, due to shorter internode lengths; shorter broader leaves; less branching of the tassels; and the development of anthers on the normally pistillate ear, resulting in perfect flowers (Emerson and Emerson, 1922).
In maize and probably other plant species, the reduced stature is primarily the result of a decrease in the final length of shoot cells. A reduction in the number of cells per internode is also a factor. Although GA deficiency affects maize shoot and mesocotyl cell length, coleoptile cell lengths are unaffected, suggesting that coleoptile cell extension is independent of GA. The reduced plant height of GA deficient/responsive mutants of maize is a characteristic common to GA deficient/responsive mutants from a number of plant species including Arabidopsis, tomato, rice, pea, and barley. Interestingly, the reduced height phenotype appears to be more responsive to GA levels than the development of anthers on the ear. This is true because, despite the semi-dwarfed to non-dwarfed stature of An1 mutants, they remain anther-eared.
Gibberellic acid levels also affect fertility in plants. For example, GA can be sprayed directly on plants to affect fertility. The nature of the effect is species specific, that is, in some species excess GA enhances fertility; whereas, in other species, GA reduces fertility. The effect depends on the reproductive mechanics of the species, and on the structure or function affected by GA.
In maize, a monecious plant with diclinous flowers, staminate flowers form on the tassel, while pistillate flowers form on the ear. Maize ears arise from axillary buds. Protuberances develop in an acropetal gradient on the ear that bifurcates-becoming two lobed. However, the diclinous nature of the mature flowers belies the fact that all flowers in the tassel and ear are initially perfect. Very early during their development, differentiation of pistillate and the staminate structures is arrested in the tassel and ear, respectively (Cheng et al., 1983). Flowers, known as florets in maize, are paired in the ear. Each pair arises from bifurcation of a spikelet, with one floret proximal to the ear axis and the other distal. Development of staminate structures in the ear is arrested in both florets, as is development of the pistillate structure in the proximal floret. Thus, the ovule of the distal floret contains the only mature gametophyte found in the ear, and when the enclosed egg and polar nucleus are fertilized, they develop as a kernel. Florets in the anther arise in a similar fashion, with development of the pistillate structures of both florets arrested very early, while stamens develop in both florets.
Reduced GA levels affect the development of pistils and stamens in maize by releasing an arrest on development of the stamens in both florets of the ear. This results in a staminate flower in the proximal floret and a mature perfect flower in the distal floret. The development of pistils and stamens in the tassel of GA deficient mutants is delayed, but otherwise is unaffected. Thus, GA is required for the normal arrested development of stamens observed in both florets of the ear. The proximal anthers on ears of GA deficient responsive mutants produce mature pollen that accumulates starch and possesses a germ pore; these are indications of a functional gametophyte. Sexual determination of tassel florets in these mutants appears to be normal, with both florets developing fertile anthers, while the pistillate structures fail to develop. The effect of these mutations on the tassels appears to be limited to reducing branching and causing a poor pollen shed apparently due to failure of the glumes to open.
In maize, tassels and shoots have served as sources for the identification of a number of GA biosynthetic intermediates (Suzuki et al., 1992; Hedden et al., 1982). In addition to being present in shoots, GAs have been shown to be present in root tips of Pisum (Coolbaugh, 1985) and in immature seeds of Pharbitis (Barendse et al., 1983).
Gibberellic acids are synthesized from the isoprenoid GGPP, beginning with the cyclizations of GGPP to CPP, then CPP to ent-kaurene, catalyzed by kaurene synthetase A and B, respectively (Duncan et al., 1981). Most higher plants are thought to be like maize in that, in maize, ent-kaurene is oxidized stepwise to 7-hydroxy-kaurenoic acid, which is converted to the first true gibberellin; GA.sub.12 -aldehyde (Suzuki et al., 1992). The latter compound then is oxidized further to an active GA by one of three parallel pathways. In maize the dominant pathway appears to be the early 13-hydroxyl pathway (Hedden et al., 1982), with GA1 being the penultimate, active product, typically present in less than 1 ug/100 gfwt amounts (Fujioka et al., 1988).
The biosynthetic block in four of the five documented GA-deficient mutants of maize has been predicted by measuring accumulation of endogenous GA biosynthetic intermediates, and measuring growth responses to, and determining the fate of, intermediates (Fujioka et al., 1988). The precise biosynthetic role of the fifth locus, An1, has remained undetermined heretofore. Mutations in An1 result in a GA-deficient phenotype, curable with applied ent-kaurene, which suggested that the An1 gene product functions in ent-kaurene synthesis.
Genes have been cloned from maize using the Mutator transposable element family (Mu) to generate gene tagged mutants. Among the genes thus cloned are al (O'Reilly et al., 1985); bz2 (McLaughlin et al., 1987); hcf106 (Marteinssen et al., 1989); hm1 (Johal et al., 1992); iojap (Han et al., 1992); vp1 (McCarty et al., 1989) and yl (Buckner et al., 1990). However, the use of the Mu system for cloning is not predictable.